Ann Surg Treat Res.  2024 Nov;107(5):252-263. 10.4174/astr.2024.107.5.252.

The efficacy of exosomes from human chemically derived hepatic progenitors in liver damage alleviation: a preclinical experimental study

• Kim M
• Kim TH
• Salas ESS
• Jeon S
• Shin JH
• Choi D

• KMID: 2560739
• DOI: http://doi.org/10.4174/astr.2024.107.5.252

Abstract

Purpose
Over the past decade, interest in exosomes as therapeutics has surged. In particular, stem-cell–derived exosomes may be more effective as a treatment for liver disease than the stem cells themselves. We have previously developed human chemically derived hepatic progenitors (hCdHs) from human hepatocytes. hCdHs can differentiate into hepatocytes and cholangiocytes, regenerating the liver in mouse models. In this study, we evaluated the mitigating effects of hCdHs-derived exosomes (hCdHs-exo) on liver damage and compared them with those of exosomes from bone marrow mesenchymal stem cells (BMMSCs-exo).
Methods
Exosomes were isolated from hCdHs and BMMSCs by culturing cells in large quantities and separating the exosomes from the culture medium using ultracentrifugation. Isolated exosomes were characterized by various methods before experimental use. In vitro, the ability of exosomes to inhibit activation of hepatic stellate cells (HSCs) by transforming growth factor beta 1 was evaluated. In vivo, exosomes were injected into mice with carbon tetrachloride (CCl₄ )-induced liver damage, and their effectiveness in mitigating liver damage was assessed by histological staining and biochemical analysis.
Results
The analyses confirmed the successful isolation of exosomes from both cell types. In vitro, hCdHs-exo significantly reduced the levels of transcription factors and activation markers in induced HSCs. In vivo, hCdHs-exo effectively alleviated liver damage caused by CCl₄ . Furthermore, both in vitro and in vivo studies confirmed that hCdHs-exo had a greater effect in alleviating liver damage than did BMMSCs-exo.
Conclusion
These results demonstrate that hCdHs-exo, similarly to hCdHs, have superior efficacy in alleviating liver damage compared with BMMSCs-exo.

Keyword

Exosomes; Hepatocytes; Human chemically derived hepatic progenitors; Liver; Mesenchymal stem cells

Figure

• 

  Fig. 1 Isolation and characterization of exosomes derived from human chemically derived hepatic progenitors (hCdHs) and bone marrow mesenchymal stem cells (BMMSCs). (A) Quantitative real-time PCR analysis of hepatic progenitor marker (CK19, EpCAM, SOX9, and CD44) and hepatocyte marker (CK18, ALB, HNF4α, CYP1A2, ASGR1, and MRP2) genes. GAPDH was used as an internal control. Data are shown as mean ± standard deviation (n = 3). Data were analyzed by 2-tailed t-tests (***P < 0.001, ****P < 0.0001). (B) Representative phase contrast (left) and double immunofluorescence (right) images of staining for the hepatic progenitor markers AFP (green)/EpCAM (red) and CK19 (green)/SOX9 (red), and for the hepatocyte markers HNF4α (green)/albumin (red), MRP2 (green)/ASGR1 (red), and CK18 (green). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 500 µm and 100 µm. (C) Brightfield imaging of hCdHs and BMMSCs. Scale bars, 500 µm. (D) Size distribution of hCdHs-derived exosomes (hCdHs-exo) and BMMSCs-derived exosomes (BMMSCs-exo) as assessed by nanoparticle tracking analysis. (E) Morphological analysis of isolated hCdHs-exo and BMMSCs-exo by cryogenic transmission electron microscopy (white arrows). Scale bar, 200 nm. (F) Expression of exosomal (CD9, CD63, and CD81) and non-exosomal (GM130 and calnexin) markers was evaluated in isolated exosome samples by flow cytometry using marker-specific antibodies. (G) Expression of exosome markers in cell lysates and exosomes were confirmed by western blotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 2 Human chemically derived hepatic progenitors-derived exosomes (hCdHs-exo) suppress hepatic stellate cell activation by transforming growth factor beta 1 (TGF-β1). (A) Confocal microscopy data were acquired after processing DiR-labeled exosomes in human hepatic stellate cells (HHSteCs). Scale bars, 10 µm. (B) Flow cytometry analysis of HHSteC treated with DiR-labeled exosomes. (C) Representative phase contrast images of HHSteCs treated with bone marrow mesenchymal stem cells-derived exosomes (BMMSCs-exo) or hCdHs-exo for 48 hours in the presence of TGF-β1 (2 ng/mL). Scale bars, 100 µm. (D) Western blot analysis of HHSteC incubated with BMMSCs-exo or hCdHs-exo for 48 hours in the presence of TGF-β1 (2 ng/mL). (E) Representative immunofluorescence images of alpha smooth muscle actin (α-SMA; green). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 100 µm. (F) Quantitative real-time PCR analysis of liver fibrosis marker genes (α-SMA, Col1α1, TGFβ1, and TIMP1). GAPDH was used as an internal control. Data are shown as the mean ± standard error of mean (n = 3 independent experiments). Data were analyzed by 2-tailed t-tests (*P < 0.05, ***P < 0.001, and ****P < 0.0001). NS, not significant.

• 

  Fig. 3 Human chemically derived hepatic progenitors-derived exosomes (hCdHs-exo) alleviate liver damage induced by carbon tetrachloride (CCl4). (A) 1, 2, and 3 hours after tail vein injection of DiR-labeled bone marrow mesenchymal stem cells-derived exosomes (BMMSCs-exo) and hCdHs-exo, in vivo tracking was confirmed using an in vivo smart imaging system. (B) Diagram of the experimental plan. (C) Body weight changes during treatment with CCl4 via intraperitoneal injection and with phosphate-buffered saline (PBS), BMMSCs-exo, and hCdHs-exo via tail vein injection. (D) ALT and AST levels analyzed by collecting blood from sacrificed mice. Each value is the mean ± standard error of mean (SEM) (n = 3 independent experiments). (E) Representative photographs of livers from mice in each group. Histopathologic features of liver sections from animals with CCl4-induced liver damage were evaluated using H&E, Masson’s trichrome, Sirius red, and immunohistochemistry staining (IHC; alpha smooth muscle actin [α-SMA]). Scale bars, 100 µm. Liver histopathology grading was evaluated by an Ishak (modified Knodell) scoring system after treatment. Data are expressed as the mean ± SEM (n = 3–5; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). NS, not significant.

Reference

1. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002; 2:569–579. PMID: 12154376.

2. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012; 64:676–705. PMID: 22722893.

3. Théry C. Exosomes: secreted vesicles and intercellular communications. F1000 Biol Rep. 2011; 3:15. PMID: 21876726.

4. Kalluri R. The biology and function of exosomes in cancer. J Clin Invest. 2016; 126:1208–1215. PMID: 27035812.

5. Nazimek K, Bryniarski K, Santocki M, Ptak W. Exosomes as mediators of intercellular communication: clinical implications. Pol Arch Med Wewn. 2015; 125:370–380. PMID: 25978300.

6. Aheget H, Mazini L, Martin F, Belqat B, Marchal JA, Benabdellah K. Exosomes: their role in pathogenesis, diagnosis and treatment of diseases. Cancers (Basel). 2020; 13:84. PMID: 33396739.

7. Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta. 2012; 1820:940–948. PMID: 22503788.

8. Gutierrez-Millan C, Calvo Díaz C, Lanao JM, Colino CI. Advances in exosomes-based drug delivery systems. Macromol Biosci. 2021; 21:e2000269. PMID: 33094544.

9. Joo HS, Suh JH, Lee HJ, Bang ES, Lee JM. Current knowledge and future perspectives on mesenchymal stem cell-derived exosomes as a new therapeutic agent. Int J Mol Sci. 2020; 21:727. PMID: 31979113.

10. Mahla RS. Stem cells applications in regenerative medicine and disease therapeutics. Int J Cell Biol. 2016; 2016:6940283. PMID: 27516776.

11. Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. Stem cell therapy: use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science. 2014; 345:1247391. PMID: 25146295.

12. Trounson A, McDonald C. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell. 2015; 17:11–22. PMID: 26140604.

13. Balistreri CR, De Falco E, Bordin A, Maslova O, Koliada A, Vaiserman A. Stem cell therapy: old challenges and new solutions. Mol Biol Rep. 2020; 47:3117–3131. PMID: 32128709.

14. Nikfarjam S, Rezaie J, Zolbanin NM, Jafari R. Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine. J Transl Med. 2020; 18:449. PMID: 33246476.

15. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014; 15:4142–4157. PMID: 24608926.

16. Hade MD, Suire CN, Suo Z. Mesenchymal stem cell-derived exosomes: applications in regenerative medicine. Cells. 2021; 10:1959. PMID: 34440728.

17. Gołębiewska JE, Wardowska A, Pietrowska M, Wojakowska A, Dębska-Ślizień A. Small extracellular vesicles in transplant rejection. Cells. 2021; 10:2989. PMID: 34831212.

18. Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med. 2017; 49:e346. PMID: 28620221.

19. Maqsood M, Kang M, Wu X, Chen J, Teng L, Qiu L. Adult mesenchymal stem cells and their exosomes: sources, characteristics, and application in regenerative medicine. Life Sci. 2020; 256:118002. PMID: 32585248.

20. Kim Y, Kang K, Lee SB, Seo D, Yoon S, Kim SJ, et al. Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells. J Hepatol. 2019; 70:97–107. PMID: 30240598.

21. Salas-Silva S, Kim Y, Kim TH, Kim M, Seo D, Choi J, et al. Human chemically-derived hepatic progenitors (hCdHs) as a source of liver organoid generation: application in regenerative medicine, disease modeling, and toxicology testing. Biomaterials. 2023; 303:122360. PMID: 38465578.

22. Kim Y, Kim YW, Lee SB, Kang K, Yoon S, Choi D, et al. Hepatic patch by stacking patient-specific liver progenitor cell sheets formed on multiscale electrospun fibers promotes regenerative therapy for liver injury. Biomaterials. 2021; 274:120899. PMID: 34034028.

23. Kim M, Kim Y, Silva ES, Adisasmita M, Kim KS, Jung YK, et al. Enhancing generation efficiency of liver organoids in a collagen scaffold using human chemically derived hepatic progenitors. Ann Hepatobiliary Pancreat Surg. 2023; 27:342–349. PMID: 37661098.

24. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995; 22:696–699. PMID: 7560864.

25. Kim J, Lee C, Shin Y, Wang S, Han J, Kim M, et al. sEVs from tonsil-derived mesenchymal stromal cells alleviate activation of hepatic stellate cells and liver fibrosis through miR-486-5p. Mol Ther. 2021; 29:1471–1486. PMID: 33348053.

26. Campana L, Iredale JP. Regression of liver fibrosis. Semin Liver Dis. 2017; 37:1–10. PMID: 28201843.